U.S. patent application number 10/808503 was filed with the patent office on 2005-09-29 for lactobacillus paracasei strain gm-080 for treating allergy related diseases.
This patent application is currently assigned to GenMont Biotech Inc.. Invention is credited to Chang, Tzu-Chi, Hsu, Ching-Hsiang, Lai, Cheng-Wei, Su, Wei-Chih, Wang, Ying-Yu.
Application Number | 20050214271 10/808503 |
Document ID | / |
Family ID | 34990121 |
Filed Date | 2005-09-29 |
United States Patent
Application |
20050214271 |
Kind Code |
A1 |
Hsu, Ching-Hsiang ; et
al. |
September 29, 2005 |
LACTOBACILLUS PARACASEI STRAIN GM-080 FOR TREATING ALLERGY RELATED
DISEASES
Abstract
The present invention provides an isolated microorganism strain,
Lactobacillus paracasei GM-080, which is found to be effective in
treating allergy. The use of the Lactobacillus paracasei GM-080 in
treating allergy related disease is also provided.
Inventors: |
Hsu, Ching-Hsiang; (Tainan
County, TW) ; Su, Wei-Chih; (Tainan County, TW)
; Wang, Ying-Yu; (Tainan County, TW) ; Chang,
Tzu-Chi; (Tainan County, TW) ; Lai, Cheng-Wei;
(Tainan County, TW) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Assignee: |
GenMont Biotech Inc.
Tainan County
TW
|
Family ID: |
34990121 |
Appl. No.: |
10/808503 |
Filed: |
March 25, 2004 |
Current U.S.
Class: |
424/93.45 ;
435/252.9 |
Current CPC
Class: |
C12N 1/20 20130101; Y10S
435/853 20130101; C12N 1/205 20210501; C12R 2001/225 20210501 |
Class at
Publication: |
424/093.45 ;
435/252.9 |
International
Class: |
A61K 045/00; C12N
001/20 |
Claims
In the claims:
1. A biologically pure microorganism, Lactobacillus paracasei
GM-080, deposited at the China Center for Type Culture Collection
under CCTCC No.: CCTCC M 204012.
2. A composition comprising the biologically pure microorganism
according to claim 1.
3. The method according to claim 14, wherein the allergy related
disease is eczema.
4. The method according to claim 14, wherein the allergy related
disease is atopic dermatitis.
5. The method according to claim 14, wherein the allergy related
disease is allergic conjunctivitis.
6. The method according to Claim 14, wherein the allergy related
disease is asthma.
7. The method according to claim 14, wherein the allergy related
disease is rhinitis.
8. The composition according to claim 2, wherein the microorganism
is live.
9. The composition according to claim 2, wherein the microorganism
is inactivated.
10. The composition according to claim 2 which is in the form of a
pharmaceutical composition, dietary supplement, food, or a
component thereof.
11. A method for stimulating IFN-.gamma. secretion in a subject
comprising administering to said subject a composition comprising
the biologically pure microorganism according to claim 1.
12. The method according to claim 11, wherein the microorganism is
live.
13. The method according to claim 11, wherein the microorganism is
inactivated.
14. A method for treating an allergy related disease in a subject
comprising administering to said subject a composition comprising
the biologically pure microorganism according to claim 1.
15. The method according to claim 14, wherein the allergy related
disease is selected from the group consisting of airway
hyperreactivity and inflammation, atopic dermatitis, allergic
conjunctivitis, rhinitis, sinusitis, hypersensitive pneumonia,
extrinsic allergic alveolitis, urticaria, eczema, anaphylaxis,
angioedema, allergic and migraine headache, certain
gastrointestinal disorders, and asthma.
16. The method according to claim 15, wherein the allergy related
disease is airway hyperreactivity or inflammation.
17. The method according to claim 14, wherein the allergy related
disease is associated with exposure to aeroallergen.
18. The method according to claim 14, wherein the microorganism is
live.
19. An in vitro culture comprising Lactobacillus paracasei GM-080,
deposited at the China Center for Type Culture Collection under
CCTCC No.: CCTCC M 204012.
20. An in vitro culture comprising Lactobacillus paracasei GM-080,
deposited at the China Center for Type Culture Collection under
CCTCC No.: CCTCC M 204012 and one or more cells selected from the
group consisting of spelenocytes and peripheral blood mononuclear
cells.
21. The method according to claim 14, wherein the microorganism is
inactivated.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the invention
[0002] The invention mainly relates to a novel microorganism strain
Lactobacillus paracasei GM-080 and its use for stimulating
IFN-.gamma. secretion and treating allergy related diseases.
[0003] 2.Description of the Related Art
[0004] Allergy refers to an acquired potential to develop
immunologically mediated adverse reaction to normally innocuous
substances. Allergic reaction provokes symptoms such as itching,
coughing, wheezing, sneezing, watery eyes, inflammation and
fatigue. It is normally believed that allergic reaction includes an
early specific immune response and a late inflammatory reaction. It
is reported that allergens (e.g., pollens and mite dust) mediate
the early phase of allergy by stimulating high affinity
immunoglobulin (IgE) receptors. For instance, mast cells and
basophils, when stimulated by allergens, will release histamine and
cytokines. The cytokines released from mast cells and basophils
then mediate the late phase of allergy by recruiting inflammatory
cells. It is also reported that the influx of eosinophils,
macrophages, lymphocytes, neutrophils and platelets starts the
vicious inflammatory cycle. This late phase of allergy amplifies
the initial immune response, which in turn triggers the release of
more inflammatory cells (Blease et al. Chemokines and their role in
airway hyper-reactivity. Respir Res 2000;1:54-61).
[0005] Various therapies have been pursued in order to treat the
symptoms of allergies. Among them, anti-allergics and histamine
H-receptor antagonists (anti-histamines) have been used. Histamine
antagonists are administered to antagonize the action of histamine
released from mast cells in response to the presence of allergens.
They reduce the redness, itching and swelling caused by the action
of histamine on the target tissues, and serve to prevent or
alleviate many of the symptoms resulting from degranulation of mast
cells. However, anti-histamines have also been associated with
adverse reactions such as diminished alertness, slowed reaction
times and somnolence (U.S. Pat. No. 6,225,332).
[0006] There are also some reports on the treatment of allergies by
regulating cytokines. Among them, interferon-.gamma. (IFN-.gamma.)
was found to inhibit the over-expression of cytokines in Th2
lymphocytes, especially the secretion of IL-4 to lower the
proliferation of B cells. Also, IFN-.gamma. could stimulate the
immune response of Th1 and repress the synthesis of IgE (Sareneva T
et al. Influenza A virus-induced IFN-.alpha./.beta. and IL-18
synergistically enhance IFN-.gamma. gene expression in human T
cells. J Immunol 1998; 160:6032-6038; Shida K et al. Lactobacillus
casei inhibits antigen-induced IgE secretion through regulation of
cytokine production in murine splenocyte cultures. Int Arch Allergy
Immunol 1998;1 15:278-287). Since IFN-.gamma. can repress B cell
proliferation and IgE secretion, it is believed that IFN-.gamma. is
effective in treating allergy.
[0007] Lactic acid bacteria, which are gram-positive bacteria, are
commonly used in industrial food fermentations. In recent studies,
lactic acid bacteria were shown to stimulate IFN-.gamma. secretion
of cells (Contractor NV et al. Lymphoid hyperplasia, autoimmunity
and compromised intestinal intraepithelial lymphocyte development
in colitis-free gnotobiotic IL-2-deficient mice. J Immunol 1998;
160:385-394). Some specific lactic acid bacteria, such as
Bifidobacterium lactis and Lactobacillus brevis subsp., were found
to stimulate IFN-.gamma. secretion of lymphocytes in blood derived
from mice and humans (U.S. patent Publication No. US 2002/0031503
A1; U.S. Pat. No. 5,556,785). It was also reported that lactic acid
bacteria could stimulate lymphocytes derived from humans or mice to
secrete Interleukin-12 (IL-12), which was a T cell stimulatory
cytokine activating T cells and NK cells to secrete IFN-.gamma.
(Hessle et al. Lactobacilli from human gastrointestinal mucosa are
strong stimulators of IL-12 production. Clin Exp Immunol 1999;
116:276-282).
[0008] Lactobacillus paracasei has been used for manufacturing
Cheddar and Italian ewe cheeses for a long time. It was found to
grow and sustain high viability in cheese during ripening
(Gardiner, G., Ross, R. P., Collins, J. K., Fitzgerald, G.,
Stanton, C. Development of a probiotic cheddar cheese containing
human-derived Lactobacillus paracasei strains. Appl Environ
Microbiol. 1998; 64: 2192-2199; Angelis, M., Corsetti, A., Tosti,
N., Rossi, J., Corbo, M. R., Gobbetti, M. Characterization of
non-starter lactic acid bacteria from Italian ewe cheeses based on
phenotypic, genotypic, and cell wall protein analyses. Appl Environ
Microbiol. 2001; 67: 2011-2020). L. paracasei was noticed to
produce anti-bacteria and anti-yeast compounds such as
H.sub.2O.sub.2 and proteinaceous active substance in human vagina
and oral cavity (Atanassova, M., Choiset, Y., Dalgalarrondo, M.,
Chobert, J.-M., Dousset, X., Ivanova, I., Haertk, T. Isolation and
partial biochemical characterization of a proteinaceous
anti-bacteria and anti-yeast compond produced by Lactobacillus
paracasei subsp. paracasei strain M3. Int. J Food Microibiol. 2003;
87: 63-73; Ocaia, V. S., Holgado, A. A. P. de R., Nader-Macas, M.
E. Growth inhibition of Staphylococcus aureus by
H.sub.2O.sub.2-producing Lactobacillus paracasei subsp. paracasei
isolated from the human vagina. FEMS Immunol. Med. Microbiol. 1999;
23: 87-92; Sookkhee S., Chulasiri, M., Prachyabrued, W. Lactic acid
bacterial form healthy oral cavity of Thai volunteers: inhibition
of oral pathogens. Journal of Applied Microbiology 2001;
90:172-179).
SUMMARY OF THE INVENTION
[0009] The invention provides a novel microorganism strain
Lactobacillus paracasei GM-080.
[0010] In another aspect, the invention provides a composition
comprising the microorganism strain Lactobacillus paracasei
GM-080.
[0011] In another aspect, the invention provides a method for
treating allergy related diseases in a subject comprising
administering said subject with a composition comprising the
microorganism strain Lactobacillus paracasei GM-080; wherein the
complication is preferably selected from the group consisting of
airway hyperreactivity and inflammation, atopic dermatitis,
allergic conjunctivitis, rhinitis, sinusitis, hypersensitive
pneumonia, extrinsic allergic alveolitis, urticaria, eczema,
anaphylaxis, angioedema, allergic and migraine headache, certain
gastrointestinal disorders, and asthma.
[0012] In still another aspect, the invention provides a method for
stimulating IFN-.gamma. secretion in a subject comprising
administering said subject with a composition comprising the
microorganism strain Lactobacillus paracasei GM-080.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 illustrates the 1000 X microscopic view of GM-080
subjected to Gram stain.
[0014] FIG. 2 illustrates the results of agarose gel analysis of
16s rDNA fragments amplified by PCR of GM-080 and lactic acid
bacterial strains CCRC12913, CCRC14001 and CCRC16100; M represents
molecular marker; 1 represents GM-080; 2 represents CCRC12913; 3
represents CCRC 14001; and 4 represents CCRC 16100.
[0015] FIG. 3 illustrates the 16s rDNA sequence alignment of GM-080
and lactic acid bacterial strains CCRC12913, CCRC14001, CCRC16100,
KLB58, PB4, and F31.
[0016] FIG. 4 illustrates a 16s rDNA phylogenetic distance tree
comparing GM-080 of the invention with related lactic acid
bacteria.
[0017] FIG. 5 illustrates the RAPD analysis of GM-080 and the
conventional lactic acid bacterial strains; M: 100-bp ladders, Lane
1: GM-080; Lane 2: Lactobacillus paracasei ATCC 25598; Lane 3:
Lactobacillus paracasei ATCC 25302; Lane 4: Lactobacillus paracasei
ATCC 335; Lane 5: Lactobacillus paracasei ATCC 11582; Lane 6:
Lactobacillus paracasei ATCC 27216.
[0018] FIG. 6 illustrates the SDS-PAGE patterns of the cell wall
proteins of GM-080, conventional Lactobacillus paracasei and
Lactobacillus fermentum strains; wherein M represents protein
molecular weight; Lane 1 represents Lactobacillus paracasei; Lane 2
represents Lactobacillus paracasei GM-080; Lane 3 represents
Lactobacillus fermentum; F1 represents a specific band of
Lactobacillus fermentum; and P1, P2 and P3 represent specific bands
of Lactobacillus paracasei.
[0019] FIG. 7 illustrates Der p 5 specific IgG (white bars) and IgE
(black bars) levels in serum of Der p 5-sensitized BALB/c mice
challenged with inhalation Der p 5; A represents the group treated
with MRS broth; B represents the group treated with L. casei; and C
represents the group treated with GM-080.
[0020] FIG. 8 illustrates the cell counts of macrophage, lymphocyte
and eosinophil in the brochoalveolar lavage of Der p 5-sensitized
mice; A represents the group treated with MRS broth; B represents
the group treated with L. casei; and C represents the group treated
with GM-080.
[0021] FIG. 9 illustrates the IFN-.gamma. secretion in the
brochoalveolar lavage of Der p 5-sensitized mice; A represents the
group treated with MRS broth; B represents the group treated with
L. casei; and C represents the group treated with GM-080.
[0022] FIG. 10 illustrates the effect of inactive GM-080 on IgE
production in Der p5-sensitized BALB/c mice. Der p5-sensitized mice
were orally administered with different dosage of GM-080 or
distilled water (control) per day for three weeks. The levels of
serum Der p5-specific IgE were determined by ELISA. While comparing
with the control group, *(p<0.1) and **(p<0.05) are
significantly different by Kruskal-Wallis H test and the posteriori
comparison was used by the Dunnett t Test.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention provides a novel microorganism strain
Lactobacillus paracasei GM-080, which is capable of treating
allergy. The strain GM-080 was deposited with the China Center for
Type Culture Collection (CCTCC) under the accession number of CCTCC
M 204012 on Feb. 19, 2004.
[0024] The Lactobacillus paracasei GM-080 is isolated from the
healthy human GI tract. A tissue sample taken from the stomach,
intestine or duodenum is suspended in a MRS broth medium containing
100 .mu.g/mL ampicillin cultured at 37.degree. C. for 2 days and
then streak plating on agar plates. The lactic acid bacterial
colonies growing on the plates can be preliminarily screened under
a microscopy examination. Candidate strains are then co-cultured
with splenocytes. The amount of IFN-.gamma. thus produced by
splenocytes in the broth is determined. Then, GM-080 is selected
for its high productivities of IFN-.gamma..
[0025] The mycological characteristics of the GM-080 are shown
below:
[0026] (a) Morphological Characteristics:
[0027] (1) Shape and size of cell: bacillus, which has a rod-like
shape with round edge when the cells after cultured at 37.degree.
C. overnight in MRS broth were observed with a microscope.
[0028] (2) Motility: motile
[0029] (3) Flagella: none
[0030] (4) Sporulation: no spore-forming
[0031] (5) Gram-stain: positive
[0032] (b) Cultural Characteristics:
[0033] (1) Medium: MRS broth (DIFCO.RTM. 0881), final pH
6.5.+-.0.2
[0034] (2) Cultural condition: 37.degree. C. anaerobic or aerobic
culture
[0035] (3) Antibiotic resistance: Ampicillin 100 .mu.g/mL
[0036] (c) Physiological Characteristics:
[0037] (1) Catalase: positive
[0038] (2) Oxidase: negative
[0039] (3) API 50 CHL test: API 50 CHL system is used for
identification of lactic acid bacteria. By assaying the responses
of a serious of enzymes, the characters of the lactic acid are
established. The result of API 50 CHL test of GM-080 is listed in
Table 1:
1TABLE 1 Reference: GM-080 VERY GOOD IDENTIFICATION TO THE GENUS
Strip: API 50CHL Profile: -----+---- +++++---++ --+-++++++
-++------+ +-+----+-- 0 - GLY - ERY - DARA - LARA - RIB + DXYL -
LXYL - ADO - MDX - GAL + GLU + FRU + MNE + SBE + RHA - DUL - INO -
MAN + SOR + MDM - MDG - NAG + AMY - ARB + ESC + SAL + CEL + MAL +
LAC + MEL - SAC + TRE + INU - MLZ - RAF - AMD - GLYG - XLT - GEN +
TUR + LYX - TAG + DFUC - LFUC - DARL - LARL - GNT + 2 KG - 5 KG - -
- - - - Significant taxa - - - % Id. - T - - - Tests against - - -
- - Lacto.para.paracasei 1 94.9 0.74 2 Lacto.para.paracasei 3 5.0
0.59 5 Next choice Lacto.rhamnosus 0.1 0.39 4 Lacto.para.paracasei
1:2 test(s) against AMYGDALINE (AMY) 98% MELEZITOSE (MLZ) 93%
Lacto.para.paracasei 3:5 test(s) against L--SORBOSE (SBE) 20%
D-SORBITOL (SOR) 20% AMYGDALINE (AMY) 99% D-TURANOSE (TUR) 20%
GLUCONATE (GNT) 20% Next choice Lacto.rhamnosus :4 test(s) against
L-RHAMNOSE (RHA) 100% METHYL-D-CLUCOSIDE (MDG) 85% AMYGDALINE (AMY)
99% MELEZITOSE (MLZ) 99%
[0040] (d) Genetic Characteristics:
[0041] 16S rDNA sequence analysis of GM-080 is determined. The
result shows that GM-080 is highly homologous to other
Lactobacillus paracasei strains (as shown in FIG. 2). Moreover, the
phylogenetic distance tree is shown in FIG. 3. Also, randomly
amplified polymorphic DNA (RAPD analysis) was performed. It shows
that GM-080 belongs to Lactobacillus paracasei, but has a specific
16S rDNA sequence. Given the above, GM-080 is a novel Lactobacillus
paracasei strain.
[0042] (e) Cell wall proteins of GM-080:
[0043] The cell wall proteins of GM-080 show similar pattern when
compared with other conventional Lactobacillus paracasei strains.
The SDS-PAGE patterns of the cell wall proteins of GM-080 are shown
in FIG. 4.
[0044] (f) Standardized detection system for identifying
GM-080:
[0045] The standard detection system for identifying microorganism
is disclosed in U.S. patent application Ser. No. 10/446,781, filed
on May 29, 2003, using gene expression difference of a test cell
line culturing with and without a given microorganism as a marker
for identification. The genes tested are listed in Table 2.
2TABLE 2 Gene Gene Gene Gene FHR-4 FGF19 FKBP1B-a FGF20 FGF13-c
FGF10 FGF14 FGF11 FGF5-b FGF1-a FGF6 FGF1-b FCGBP FCAR-f FCGR1A
FCAR-g FADD ELK3 FCAR-a ENG ELA2 CXCR4 EGR1 CXCL16 CX3CR1 CSF2RB
CXCL1 CSF3R-a CRL3 COL3A1 CRTAM CR1 CMRF-35H CHUK CNR1-a CKTSF1B1
CDC25A CD163 CDH3 CD164 CD97-b CD81 CD109 CD83 CD79A-a CD58 CD79A-b
CD59 CD37 CD22 CD38 CD24 CD7 CD3G CD8A CD3Z CD2-a CCRL2 CD2-b CD1A
CCR4 CCL25 CCR5 CCL26 CCL19 CCL8 CCL20 CCL11 CAMK4 C9 CCBP2 CABIN1
C5 C1S C6 C2 C1QTNF2 BTNL2 C1QTNF3 BY55 BLR1-b BCL2-a BLR1-c BCL2-b
AP1S1-b ALDH1A1 AP1S1-c AOAH ADRB2 ACVR1B-c ATF2-a ACVR1B-d
FKBP1B-b FGF21 FLJ14639 FGF22 FGF16 FGF12-a FGF17 FGF12-b FGF7 FGF2
FGF8-a FGF3 FCGR2A FCAR-h FCGR2B FCER1A FCAR-b EP300 FCAR-c EPO
EGR2 CXCR3 EGR3 CYSLTR1 CXCL10 CSF3R-b CXCL13 CTLA1 CSNK2A1 CR2
CSNK2B CREB1-a CNR1-b CIAS1 CPA3 CIS4 CDKN1A CD200R CDKN2B-a CD209
CD151-a CD84 CD151-b CD84-H1 CD79B-a CD63 CD79B-b CD68 CD44 CD33
CD47 CD34-a CD8B1 CD4 CD9 CD5 CD2AP CD1B CD2BP2 CD1C CCR6 CCL27
CCR8 CCL28 CCL21 CCL13 CCL23-a CCL16 CCL1 CALM1 CCL2 CALM2 C7 C3
C8A C3AR1 C1QTNF4 C1QA C1QTNF6 C1QB BMPR1A BCL2-c BMPR1B BCL3 AP1S2
AMH ATF2-b AMHR2 AGT ACVR2 AIF1-a ACVR2B ACHE-b ACE-a ACVR1 ACE-b
FOG2 FGF23 FOS FHOD2 FGF18-a FGF13-a FGF18-b FGF13-b FGF8-b FGF4
FGF9 FGF5-a FCGR3A FCER1G FCGRT FCER2 FCAR-d ETEA FCAR-e EPX EGR4
DAF ELK1 E48 CXCL5 CTLA4 CXCL6 CTRP5 CSF1R CREB1-b CSF2RA CREBBP
COL1A1 CMA1 COL1A2 CMRF35 CDKN2B-b CD209L CER1 CD244 CD151-c CD86-a
CD151-d CD97-a CD79B-c CD72 CD80-a CD74 CD48 CD34-b CD53 CD36 CD14
CD5L CD19 CD6 CD3D CD1D CD3E CD1E CCR9-a CCR1 CCR9-b CCR3 CCL23-b
CCL17 CCL24 CCL18 CCL5 CALM3 CCL7 CAMK2B C8B C4BPA C8G C4BPB
C1QTNF7 C1QBP C1R C1QR1 BMPR2-a BF BMPR2-b BLR1-a BAD-a ANXA3 BAD-b
AP1S1-a AIF1-b ACVRL1 ALDH1A2 ADRB1 ACVR1B-a ACE2 ACVR2B-b ACHE-a
NCAM2 MUC4-c NCF2 MYC MORF MIF MUC1 MMD MEF2B MAPK14-a MEF2D
MAPK14-b MAPK8 MAP3K14 MAPK9 MAP3K7-a MAF MADH3 MAP2K7-a MADH4 LY6H
LY6E LY75 LY6G5B LTB-b LLT1 LTBR LTB4R-a LOC163702 LOC139429
LOC201595 LOC145314 LILRB5 LILRA2 LOC122687 LILRA3 KPNA5 JAK3 KPNB3
JUN ITGB1-a ITGA10 ITGB1-b ITGA11 ITGA3-b IRF6 ITGA4 IRF7 IRAK3
ILF2 IRAK4 ILF3-a IL19 IL-17RE-b IL20 IL-17RE-c IL-17RC-b IL16
IL-17RC-c IL17 IL11 IL3RA IL11RA-a IL4I1 IRAK2-a IGSF6 IL1F8 IGSF8
IGFBP3 IFNW1 IGLL1 IFRD1 IFNA4 IFIT2 IFNA8 IFIT4 IFI16 ICOS IFI27
ICAM3 HCGIX GPR84 HF1 GRLF1 GDF10 FOSL1 GBP2 FOSL2 NFAT5-b ITGB3
NFAT5-c ITGB3BP NCF4-a MYD88 NCF4-b MYF5 MUC2 MME-a MUC3B MME-b
MHCBFB MCP-a MHC2TA MCP-b MAPK10-a MAP3K7-b MAPK10-b MAP3K7-c
MAP2K7-b MADH5 MAP3K1 MADH6 LY9 LY6G5C LYL1 LY6G6C LTB4R-b LTB4R2-a
LTB4R2-b LAG3-b LOC205360 LOC145355 LOC221937 LOC145497 LOC128342
LILRB1 LOC136520 LILRB2 LAG3-a JUNB LAT JUND ITGBL1 ITGB4 ITK
ITGB4BP ITGB1-c ITGAE ITGB1-d ITGAL ITGA5 IRTA1 ITGA6 IRTA2 IRF2
ILF3-b IRF3 ILF3-c IL21 IL-17RE-d IL21R IL-17RE-e IL-17RC-d IL17C
IL-L7RC-e IL17F IL11RA-b IL7 IL11RA-c IL8 IL1F7 IGSF9 IL2RA IKBKB
IGSF1 IFRD2 IGSF2 IGBP1 IFNAR1 IFITM1 IFNAR2 IFNA14 IFI30 ICAM4-a
IFI35 ICAM4-b HM74 GSCL HOXA1-a GSK3A GFI1 FST GPR2 FY NFAT5-d
ITGB7-a NFATC1 ITGB8 NCF4-c NBL1 NFAT5-a NCAM1 MUC4-a MMEL2 MUC4-b
MMP9 MICA MCP-c MICB MEF2A MAPK10-c MAP3K7-d MAPK10-d MAPK3 MAP3K2
MADH7 MAP3K7IP1 MADH9 MADH1 LY6G6D MADH2 LY6G6E LY117 LTA LY64
LTB-a LOC221938 LOC147137 LEP-b LOC149620 LOC136531 LILRB3
LOC136535 LILRB4 LEP-a KITLG-a LILRA1 KITLG-b IVL ITGB5 JAK2 ITGB6
ITGB2 ITGAM ITGB1BP2 ITGAV ITGA7 ITGA2 ITGA8 ITGA3-a IRF5-a IRAK1
IRF5-b IRAK2-b IL22R IL18BP IL-23R IL18R1 IL-17RC-f IL17R IL-17RE-a
IL-17RC-a IL14 IL8RA IL15RA IL8RB IL2RB IKBKG IL2RG IKKE IGSF3
IGHMBP2 IGSF4 IGF1 IFNGR1 IFNA2 IFNGR2 IFNA21 IEI44 ICAM5 IFIT1 IF
HOXA1-b GSK3B HRAS HCC-4 GPR31 GATA1 GPR44 GATA6 IL5 IL1R1 IFNA1
IL6ST IL10RB IL10RA ICAM1 IL13RA2 GATA3 IL1B IL10 IL2 MIP-A
LOC126133 Uricase HNF4A LOC161823 PGK1 G6PT1 NT5C1A DHFR PPARG-b
PGK2 LOC200895 LOC132198 XDH PPARG-a GDA TCF2-a SLC22A12-a TCF2-b
SLC22A12-b ALDH2 PRPSAP2 MTHFR VLDLR LOC205855 YY1 NP PPAT VAV3
TRPV6-c VEGF TSA1902 TRAF4-a TRAF1 TRAF4-b TRAF2-a TNFRSF7 TNFSF5
TNFRSF8-a TNFSF6 TLR10 TLR6 TNFAIP3 TLR7 TLR3 TGIF-b TLR4-a TGIF-c
TGFB2 TBX21 TGFB3 TCF8 STAT2 SOCS5-a STAT3 SOCS5-b SERPING1 SEMA4B
SFN SEMA4C SE20-4 RPL13A SEMA3A RUNX1 REL PRL RELA PTGER2 PLAU
PECAM1 PPP3CB PFC P2RX7 NOS2A-b PAK1 NPPB NFKBIB NFATC2 NFKBIE
NFATC3 IL5RA IL1R2 None IL9-a STAT1-c STAT1-b ITGB7-b CCR2-c
IL13RA1 CCR2-a IL18 CD69 TGFB1 IL27 CD28 IL1A VCAM1-b JAK1 TNF-b
CSF3 IL6R STAT1-a IL12RB2 IL15 15MD2 HNF-1B GBP1 15MD-1 LOC169330
S100A8 IMPDH1 S100A9 MTHFD2 HDLBP G6PC LRP8 PRPS2 HPRT1 PRPSAP1
APRT XCL1 TSC22 XCR1 TYK2 TRAF5 TRAF2-b TRAF6 TRAF2-c TNFRSF8-b
TNFRSF11A TNFRSF9 TNFRSF1A TNFSF11-a TLR8-a TNFSF11-b TLR8-b TLR4-b
TH1L TLR4-c TIMP1 TGFBR1 TCP10 TGFBR2 TDGF1 STAT4 SOCS4 STATI2
SSI-1 SIVA-a SEMA4D SIVA-b SEMA4F SEMA3B RUNX2 SEMA3C SCYA3 RELB
PTPRC-a RIPK1 PTPRC-b PPP3CC PIGR PPP3R1 PILR(ALPHA) PDE4B NUP214-a
PDGFB-a NUP214-b NFKBIL1 NFATC4 NFKBIL2 NFKB1 CSF1 IL9-b CD80-b
IL13 CCR2-b CD86 IL4 IFNB1 CEBPB TIM3 IRF1 IL4R TP53 IL12B TNF-a
SERPINA3 VCAM1-a SCYA4 CCR7 IL12A IL12RB1 CSF2 ADSS STAT6 IMPDH2
IL6 LGALS9 IFNG UMOD PTGS2 LOC223071 TCF2-c PRPS1 APOE ZNF144 APOB
XPO5 ADA TRPV6-b DPP4 TRPV6-a VAV1 TPSD1 VAV2 RSF21 TRAF3-a TNFSF4
TRAF3-b TNFSF11-c TNFRSF1B TLR5 TNFRSF21 TLR4-d TLR9-a TGIF-a
TLR9-b TGFBR3 TLR1 TBXA2R TLR2 TACTILE TFCP2 SLAM TGFA SLA SSI-3
SEMA3F SUDD SEMA3E SEMA4G RNASE3 SEMA7A RNASE2 SCYE1 PRG2 SDF2
PRKG1 PTPRC-c PDPK1 RDC1 PDGFB-b PILR(BETA) NOS2A-a PIN1 NMA OPRD1
negative ORM1 ACTB NFKB2 G6PD NFKBIA
[0046] The standard detection system for identifying GM-080 takes
Jurkat cell line as a test cell line. When comparing the expression
patterns of culturing Jurkat cell line with and without GM-080, the
genes listed in Table 3 are significantly different. Furthermore,
the detection results of other Lactobacillus paracasei strains,
CCRC 12193 and CCRC 12188, are also shown in Table 3. It indicated
that these strains are all Lactobacillus paracasei, but belong to
different strains.
3 TABLE 3 Paracasei Paracasei Gene name CCRC12193 GM-080 CCRC12188
ADA ++++++ ++++ ++ BAD-a ++ +++ + BCL3 + + - BLR1-c -- + -- BMPR2-a
++ ++ + CCL2 - + - CD2AP ++ ++ + CD2-b ++ ++ + CD38 ++ ++ - CD3G
++++++ ++++++ ++++++ CD48 ++ ++ + COL1A2 -- - -- CR2 ++ ++ +
CREB1-a ++ ++ + CREB1-b +++ +++ + CX3CR1 ++ +++ ++ DAF ++ +++ +
ETEA ++ ++ + FCAR-h +++ +++++ ++ FGF23 + ++ + FHOD2 ++ ++ + HOXA1-a
+ ++ + IFNAR1 ++++ +++++ ++ IFNGR1 ++ +++ + IKKE ++ ++ - IL14 + ++
+ IL17R ++++ ++ + IL4R +++ +++ + IL7 ++ ++ + JAK1 ++ ++ + LEP-a +
+++ + LOC200895 + ++ + LY117 ++ +++ - MADH4 ++ ++ + MADH5 +++ +++ +
MAP3K14 ++ ++ + MAPK14-a ++ ++ + MAPK3 ++ +++ + MCP-a +++ +++ +
MCP-c ++ ++ + PDPK1 ++ ++ + REL ++ ++ + RIPK1 ++ ++ + SEMA3C - ++ +
TGFBR2 ++ ++ - TLR3 +++ +++ + TNFSF4 +++ +++ + TRAF3-a +++ ++ ++
TRAF6 + ++ + TSC22 +++ +++ + +: the gene expression increases in 2
folds -: the gene expression decrease in 2 folds
[0047] GM-080 is active after treating HCl solution (pH 2.0) for 3
hours and then gall for 4 hours. Therefore, GM-080 is regarded as
remaining active in digestion. GM-080 is isolated from a healthy
subject and is safe, natural, nontoxic, and meet the G.R.A.S.
(Generally Regarded as Safe) standard.
[0048] Furthermore, GM-080 strongly adhered to the epithelial cells
in the intestine. Given the above, GM-080 can stay in the intestine
for a longer time to act for modulating physiological functions.
Also, by occupying the adhesion sites of the epithelial cells in
the intestine, GM-080 bars other pathogenic bacteria from adhering
to the intestine. GM-080 is regarded as a good probiotic
bacterium.
[0049] According to the invention, GM-080 is found to stimulate
IFN-.gamma. secretion, and can be used for treating allergy related
disease.
[0050] In one aspect, the invention provides a composition
comprising GM-080. More preferably, the composition comprising
GM-080 is used for stimulating IFN-.gamma. secretion, which is
useful for treating allergy related diseases.
[0051] As used herein, the term "allergy related diseases" refers
to the diseases wherein a systematic reaction to a normal innocuous
environmental antigen, which results from the interaction between
the antigen and antibody or T cells produced by earlier exposure to
the same antigen. The term "allergic reaction" as used herein
refers to a response to innocucous environmental antigens or
allergens due to pre-existing antibody or T cells. There are
various immune mechanisms of allergic reactions, but the most
common type is the binding of allergen to IgE antibody on mast
cells that causes asthma, hay fever, and other common allergic
reactions. The allergy related diseases include airway
hyperreactivity and inflammation, atopic dermatitis, allergic
conjunctivitis, rhinitis, sinusitis, hypersensitive pneumonia,
extrinsic allergic alveolitis, urticaria, eczema, anaphylaxis,
angioedema, allergic and migraine headache, certain
gastrointestinal disorders, and asthma. According to a preferred
embodiment of the invention, the allergy related disease is airway
hyperreactivity or inflammation. In another aspect, the allergy
related disease is associated with exposure to airborne allergen
(aeroallergen) such as pollens, molds, animal dander, and
insects.
[0052] As used herein, the term "aeroallergen" is defined as having
at least the following characteristics: specific antigenic
groupings that evoke active reaginic responses, and ambient
exposure levels to which can lead to overt tissue changes in
sensitive subjects. Aeroallergens are airborne particles that can
cause respiratory, cutaneous, or conjunctival allergy. The
water-soluble portion of ragweed pollen, for example affects the
respiratory and conjunctival mucosa, and the lipid-soluble
allergens of ragweed pollen can cause a typical contact dermatitis
on exposed skin.
[0053] GM-080 is selected to have the ability to stimulate
IFN-.gamma. secretion when co-incubated with splenocytes and
peripheral blood mononuclear cells (PBMCs) in vitro. Furthermore,
in the model according to the invention, animals sensitized with an
aeroallergen and then treated with GM-080 are observed to increase
IFN-.gamma. secretion. Furthermore, the amount of aeroallergen
specific IgE is significantly lowered after treatment. On the other
hand, the amount of allergen specific IgG does not show significant
difference between before and after treatments. In addition, the
eosinophil cell count in the bronchoalveolar lavage fluids (BALF)
is enormously decreased; however, the macrophage and lymphocyte
counts in BALF are enormously increased. It evidenced that the
inflammation was relieved.
[0054] According to the invention, GM-080 for use in the treatment
of allergy can be live or inactive. Preferably, GM-080 is inactive.
For instance, the live bacterial strains can be treated with a
heating step or other treatments commonly used in the art for
killing the lactic acid bacterial as the inactive strains.
[0055] According to the invention, the lactic acid bacterial strain
can be included in a pharmaceutical composition, dietary
supplement, food, health food, medical food, or the components
thereof, which are normally administered by people. In a preferred
embodiment of the invention, the lactic acid bacterial strain can
be delivered in food form, such as in a coagulated milk product
that prepared through the fermentation of lactic acid in milk. The
food products prepared according to the invention can be
conveniently administered to infants or children.
[0056] In another aspect, the invention provides a method for
treating allergy related disease in a subject comprising
administering said subject with a composition comprising the
isolated microorganism GM-080.
[0057] In still another aspect, the invention provides a method for
stimulating IFN-.gamma. secretion in a subject comprising
administering said subject with a composition comprising the
isolated microorganism GM-080.
[0058] The following Examples are given for the purpose of
illustration only and are not intended to limit the scope of the
present invention.
EXAMPLE 1
Isolation of Lactobacillus paracasei GM-080
[0059] Sample: A piece of human stomach, intestine or duodenum
tissue taken by an endoscope was cultured in 2 mL of Lactobacillus
MRS Broth (DIFCO.RTM. 0881) containing 100 .mu.g/mL of ampicillin
for about two days at 37.degree. C. The broth was plated on MRS
agar containing CaCO.sub.3 and incubated at 37.degree. C. for two
days. Single colony growing on the plate was selected and subjected
to Gram-stain. Gram-positive bacteria were then selected. All of
the strains were cultured in Lactobacillus MRS broth at 37.degree.
C. to the stationary phase, and collected by centrifuging at 3000 g
for 15 minutes and washed with 2 mL and 1 mL PBS (phosphate
buffered saline, pH 7.2). The cultures of the strains were
re-suspended in 1 mL PBS and then heated at 95.degree. C. for 30
minutes, and were then autoclaved and stored in PBS at -20.degree.
C.
[0060] Isolation of splenocytes: Five mL blood samples derived from
healthy volunteers were added with 5 mL Ficoll-Hypaque (17-1400-02,
Pharmacia) and then centrifuged at 500 g for 30 minutes. The
splenocytes were taken. In each splenocyte sample, the cell density
was adjusted to 5.times.10.sup.6 cells per sample. The splenocyte
samples were incubated in 2 mL RPMI 1640 (pH 7.7) for 6 hours.
[0061] Isolation of peripheral blood mononuclear cells: Five mL
blood samples derived from healthy volunteers were added with 5 mL
Ficoll-Hypaque (17-1400-02, Pharmacia) and then centrifuged at 500
g for 30 minutes. The peripheral blood mononuclear cells (PBMCs)
were taken from the interface of the samples, and washed twice with
PBS. The PBMCs (10.sup.5 cells/mL) were transferred to the wells of
a six-well plate wherein each well contained 2 mL RPMI 1640 medium
of pH 7.7.
[0062] Stimulating IFN-.gamma. Secretion. The splenocyte or PBMC
samples were co-cultured with a given amount of the Gram-positive
bacteria. After the 36-hour co-culture, the cells in each sample
were collected, respectively. The collected cells were re-suspended
and centrifuged at 2000 rpm for 5 minutes. The supernatants were
taken for the determination of IFN-.gamma. level in each
sample.
[0063] Determination of IFN-.gamma. Level: IFN-.gamma. Level was
determined by ELISA, comprising the steps of:
[0064] adding 30 .mu.L of 2.5 .mu.g/mL purified mouse anti-human
IFN-.gamma. antibodies (Cat. No18181D, PharMingen.RTM., USA) in 10
mL of coating buffer (0.1 M Na.sub.2HPO.sub.4, pH 9.0) and adding
100 .mu.L of antibody solution into each well of a ELISA plate;
[0065] shaking the plate at 4.degree. C.;
[0066] washing each well of the plate with washing buffer (0.05%
Tween 20 in PBS);
[0067] adding 300 .mu.L blocking buffer (1% BSA in PBS) into each
well of the plate;
[0068] shaking the plate at room temperature for at least 2
hours;
[0069] adding 100 .mu.L of the supernatant of the splenocyte sample
to each well of the plate;
[0070] shaking the plate at 4.degree. C. overnight;
[0071] washing each well of the plate with washing buffer;
[0072] adding 150 .mu.L biotin mouse anti-human IFN-.gamma.
antibodies (Cat. No 18112D, PharMingen.RTM., USA) into each well of
the plate;
[0073] incubating the plate for 1 hour at room temperature;
[0074] washing each well of the plate with washing buffer;
[0075] adding 150 .mu.L Streptavidin-AKP diluted with dilute buffer
(1:1000) into each well of the plate;
[0076] shaking the plate for 1 hour at room temperature;
[0077] washing each well of the plate with wash buffer eight
times;
[0078] adding 200 .mu.L of substrate pNpp was added into each well
of the plate;
[0079] incubating the plates at room temperature until the
substrate reaction is completed;
[0080] measuring the absorbance of each well of the plate at 405 nm
(i.e. OD.sub.405).
[0081] Result: Among the Gram-positive bacteria, GM-080 was
selected to have the strongest ability to stimulate IFN-.gamma.
secretion in splenocyte cells and PBMCs.
EXAMPLE 2
16s rDNA Sequence Determination
[0082] DNA extraction: The genomic DNA of GM-080 and other
bacteria, CCRC12913, CCRC 14001 and CCRC 16100 were extracted using
QIAamp.RTM. DNA Stool Mini Kit (Qiagen.RTM., cat No. 51504). The
purification was performed according to the steps as listed
below:
[0083] adding 1.4 mL of ABS buffer in to the culture and vortexing
it for 1 min;
[0084] heating the solution obtained in the previous step at
70.degree. C. for 5 min;
[0085] vortexing the solution for about 15 sec and then
centrifuging it at about 13,000 rpm for 1 min;
[0086] removing the supernatant into a new centrifuge tube;
[0087] adding an InhibitEx tablet in the supernatant and shaking it
to dissolve the tablet, and then incubating at room temperature for
1 min;
[0088] centrifuging the solution at about 13,000 rpm for 3 min to
make the bacteria attach to InhibitEx;
[0089] removing the supernatant into a new centrifuge tube and then
centrifuging at about 13,000 rpm for 3 min;
[0090] taking 200.mu.L of the supernatant to a new centrifuge tube
and adding Protease K;
[0091] adding 200 .mu.L of Buffer AL and vortexing it for 15 min to
obtain a homogeneous solution;
[0092] adding 15 .mu.L of Protease K into the homogenous solution
and vortexing it for 15 sec;
[0093] incubating the solution at 70.degree. C. for 10 min;
[0094] adding 200 .mu.L of 96-100% ethanol and vortexing;
[0095] removing the solution into QIAamp spin column and
centrifuging it at about 13,000 rpm for 1 min;
[0096] removing the QIAamp spin column to a new centrifuge tube and
adding 500 .mu.L Buffer AW1, and then centrifuging it at about
13,000 rpm for 1 min;
[0097] removing the QIAamp spin column to a new centrifuge tube and
adding 500 .mu.L Buffer AW2, and then centrifuging it at about
13,000 rpm for 1 min;
[0098] removing the QIAamp spin column into a new centrifuge tube
and adding 200 .mu.L Buffer AE, and then incubating it at room
temperature for 1 min; and
[0099] centrifuging at about 13,000 rpm for 1 min to elution
DNA.
[0100] 16s rDNA fragment amplification: The primers for amplifying
L region were designed according to Lactobacillus paracasei 16S
rRNA V1 region, 5'-CAC CGA GAT TCA ACA TGG-3'(SEQ ID No. 1) and
Lactobacillus conserved 16S rRNA, 5'-CCC ACT GCT GCC TCC CGT AGG
AGT-3' (SEQ ID No. 2) (Ward, L. J. H. and Timmins, M. J. (1999)
Differentiation of Lactobacillus casei, Lactobacillus paracasei and
Lactobacillus rhamnosus by polymerase chain reaction. Lett. Appl.
Microbiol. 29: 90-92). The genomic DNA of GM-080, CCRC12913, CCRC
14001 and CCRC 16100 were taken as the template for performing PCR
reaction. The 16s rDNA PCR amplification program is as follows: (1)
95.degree. C. for 10 min; (2)95.degree. C. for 45 sec; (3)
46.degree. C. for 45 sec; (4) 72.degree. C. for 1 min; (5)
72.degree. C. for 7 min; steps 2 to 5 were repeated for 30
cycles.
[0101] 16s rDNA sequence determination: The PCR products of GM-080,
CCRC 12913, CCRC 14001 and CCRC 16100 were subjected to agarose gel
electrophoresis (FIG. 2) and sequenced. The sequences were aligned
against the multiple sequence alignment dataset (NCBI blastn,
http://www.ncbi.nlm.nih.gov/BLAST) using the ARB sequence editor
(release 8.1). It also showed that the 16s rDNA sequences of
Lactobacillus paracasei strain PB4, AY186046; F31, AF243147; KLB58,
AF243168 were similar to that of GM-080 as shown in FIG. 3
(generated with VectorNTI.TM., InforMax.phi. Inc.). In addition,
16s rDNA phylogenetic distance tree was generated with EMBL-EBI
ClustalW (http://www.ebi.ac.uk/clustalw) as shown in FIG. 4.
According to the 16S rDNA analysis, GM-080 was highly related to
Lactobacillus paracasei strain KLB58, but still distinct from
KLB58. Given the above, GM-080 belonged to Lactobacillus
paracasei.
EXAMPLE 3
Randomly Amplified Polymorphic DNA (RAPD Analysis)
[0102] DNA extraction of GM-080, Lactobacillus paracasei ATCC
25598, 25302, 335, 11582, and 27216 was performed as described in
Example 2.
[0103] The primer for random amplification was 5'-ATGTAACGCC-3'
(Gardiner, G., Ross, R. P., Collins, J. K., Fitzgerald, G.,
Stanton, C. Development of a probiotic cheddar cheese containing
human-derived Lactobacillus paracasei strains. Appl Environ
Microbiol. 1998; 64: 2192-2199).
[0104] The result of RAPD was shown in FIG. 5. According to the
RAPD analysis, GM-080 was distinct from the conventional
Lactobacillus paracasei strains. Given the above, GM-080 was a
novel Lactobacillus paracasei strain.
EXAMPLE 4
Cell Wall Proteins Extraction and Analysis of GM-080
[0105] The cell wall proteins were purified according to the method
described by Angelis (Angelis, M. D., Corsetti, A., Tosti, N.,
Rossi, J., Corbo, M. R., and Gobbetti, M. (2001) Characterization
of Non-Starter Lactic Acid Bacteria from Italian Ewe Cheeses Based
on Phenotypic, Genotypic, and Cell Wall Protein Analyses. Appl.
Environ. Microbiol. 67: 2011-2020). The cells cultured overnight in
MRS broth (Difco.RTM.) were harvested and then washed twice with
0.05 M Tris-HCl (pH 7.5) containing 0.1 M CaCl.sub.2, and
resuspended in 1 ml of the same buffer at an OD.sub.600 of 10.0.
After centrifugation at 8,000.times.g for 5 min, cell wall proteins
were extracted from the pellets with 1.0 ml of extraction buffer
(pH 8.0) containing 0.01 M EDTA, 0.01 M NaCl, and 2% (wt/vol) SDS.
Suspensions were stored at room temperature for 60 min, heated at
100.degree. C. for 5 min, and centrifuged at 11,600.times.g for 10
min at 4.degree. C. The supernatants were analyzed by 12% SDS-PAGE
and stained with Comassie blue.
[0106] The result was shown in FIG. 6. The pattern of GM-080 had
three specific bands, P1, P2 and P3 that similar to those of
Lactobacillus paracasei reported in the prior study (Angelis et al.
2001). Therefore, GM-080 was evidenced to belong to Lactobacillus
paracasei.
EXAMPLE 5
A Standardized Detection System for Identifying GM-080
[0107] Stimulation: The Jurkat cells were refreshed by adding a
fresh medium and cultured for 16 hours. Subsequently, the cells
were divided into two groups, one for the culture with the lactic
acid bacteria and the other for the culture without the lactic acid
bacteria. When the cell concentration reached 1.times.10.sup.7/10
mL, cells were stimulated for 24 h with or without 1.times.10.sup.7
different lactic acid bacteria (CCRC12193, GM-080 or CCRC12188).
After stimulation, the cells were collected, washed twice with PBS,
and used for RNA isolation.
[0108] RNA isolation and labeling: RNA was extracted from cell by
using Trizol Reagent (Life Technologies.RTM., Gaithersburg, Md.)
according to the manufacturer's instructions. 8 L of the RNA
(1long) and 2 L oligo poly-dT (12-18 mer, 1 g/L) were well mixed
and kept at 70.degree. C. for 10 minutes and then were cooled with
ice for 2 minutes. Mixed the RNA with reverse transcription
labeling mixture and 3 L Cy3-dUTP (1 mM), 2 L SuperScript III (200
U/L), and RNasin (1 L) in dark. The mixture was incubated at
50.degree. C. for 2 hours for reverse transcription, and the
reaction was terminated by adding 1.5 L 20 mM EDTA. After the
labeling, RNA was removed by NaOH treatment and neutralized by HCl.
cDNA was immediately purified with a YM30 purification kit.
[0109] Microarray fabrication: Hundreds of genes chosen were
amplified through polymerase chain reaction and quantified by
spectrophotometry at 260 nm. All purified PCR products were
adjusted to a concentration of 0.1 .mu.g/.mu.l in 50% dimethyl
sulfoxide and spotted in duplicate on UltraGAPSTM coated slides
(Corning.RTM., Inc., Corning, N.Y.). After printing, the
microarrays were UV cross-link at 700 mJoulesand stored in the
slide container in a desiccator at room temperature. The genes were
listed in Table 2 as mentioned above.
[0110] Microarray hybridization: Fluorescently labeled cDNA was
denatured in the hybridization solution (5.times.SSC, 0.1% SDS and
25% formamide) at 100.degree. C. for 5 min, cooled to ambient
temperature, and deposited onto slides. The hybridization was
carried out for 18 h at 55.degree. C. After hybridization, the
slides were successively washed in low-stringency (1.times.SSC and
0.1% SDS), medium-stringency (0.1.times.SSC and 0.1% SDS),
high-stringency (0.1.times.SSC) buffer and finally were dried by
compressed N.sub.2.
[0111] Signal detection and data analysis: N.sub.2-dried slides
were immediately scanned on a GenePix 4000B scanner (Axon
Instruments.RTM., Inc.) at the same laser power and sensitivity
level of the photomultiplier for each slide. Raw fluorescence data
were acquired (10-nm resolution), and subsequent processing and
data visualization were performed in Microsoft Excel.TM.. In order
to compare the results of independent hybridization experiments,
the local background signal was subtracted from the hybridization
signal of each separate spot, and then divided by the housekeeping
gene, .beta.-actin. The final expression of each gene was
represented in a mean of duplicates. The gene expression profiles
of the Jurkat cell cultured with and without the lactic acid
bacteria were then obtained. A group of genes upregulated or
down-regulated more than 2 fold in Jurkat cell cultured with lactic
acid bacteria (CCRC12193, GM-080 or CCRC12188) to that cultured
without the bacteria were selected. The results were shown in Table
3. The difference indicated that different species or strain can
turn on or turn off different genes of the cell. Hence, from the
gene expression profile, it indicated that CCRC12193, GM-080 and
CCRC12188 are L. paracusei but belong to different strains.
EXAMPLE 6
Adhesion of GM-080 to The Epithelial Cells in The Intestine
[0112] Caco-2 cells were taken as the epithelial cells in the
example. Caco-2 cells had functional microvilli and hydrolase
attached thereon, it exhibited differentiated morphology and
functions of a mature epithelial cell in the intestine in
vitro.
[0113] Cells: Caco-2 were cultured in Mineral essential medium
(MEM, GIBCO.RTM.) supplemented with 5% FBS at 37.degree. C. in an
5% CO.sub.2/95% air. For adhesion assay, 2 ml of monolayer of
Caco-2 cells (3.times.10.sup.5 cells/ml) were prepared on glass
cover slips that were placed in 6-well plate. The culture medium
was replaced every second day and the monolayers were used in the
adhesion assay after 2 weeks incubation. Just before use, the
monolayer was wash twice with PBS and 1.5 ml of MEM was added to
each well and incubated at 37.degree. C. for 1 h before inoculation
of bacteria.
[0114] Adhesion: 1.5 ml of (4.times.10.sup.8CFU/ml) of GM-080
washed once with PBS and resuspended in 1.5 ml MEM medium was added
to the Caco-2 cells. After 1 h of incubation at 37.degree. C.,
monolayer of cells were washed four times with PBS buffer, fixed
with 3 ml of methanol and incubated for 5 to 10 min at room
temperature, wash three times with PBS, dried in air and Gram
stained. Adherent bacteria were detected microscopically under oil
immersion (.times.100) by counting 15 random fields per coverslip
and mean.+-.SD of adhering bacteria per field was determined.
[0115] Result: After counting, there were 102.+-.23.6 GM-080
bacteria adhered to the Caco-2 cells. Therefore, GM-080 was
regarded to have strongly adhesion to Caco-2 cells according to the
standard established by Jacobsen et al. (Jacobsen, C. N., Nielsen,
R. V., Hayford, A. E., Moller, P. L., Michaelsen, K. F.,
Paerregarrd, A., Sandstrom, B., Tvede, M. and Jakobsen, M.
Screening of probiotic activities of forty-seven strains of
Lactobacillus spp. by in vitro techniques and evaluation of the
colonization ability of five selected strains in human. Appl.
Environ. Microbiol. 1999; 65: 4949-4956).
EXAMPLE 7
Activities of GM-080 and Other Lactic Acid Bacteria in An
Environment Mimicking GI Tract
[0116] Acid: The overnight-cultured GM-080, L. plantarum, L.
acidophilus, L. casei, and L. bulgaricus were added with 9 mL of
PBS with different pH values of 2.0, 2.5 and 3.2 and then further
cultured at 37.degree. C. for 3 hours. After culturing, 1 mL cells
were serially diluted with 9 mL of pH 7.4 PBS. The cell counts
before and after acid treatment were estimated and shown in Table 4
as listed below.
[0117] gall. The overnight-cultured GM-080, L. plantarum, L.
acidophilus, L. casei, and L. bulgaricus were added with 9 mL of
PBS with different pH values of 2.0 and then further cultured at
37.degree. C. for 3 hours. After culturing, the 1 mL cells were
centrifuged at 6,000 rpm for 10 min. The pellet was re-suspended
with 100 .mu.L of PBS (pH 7.2). The solution was further added with
10 mL of MRS broth containing 0.3% (w/v) of ox gall. The cells were
cultured and 1 mL of sample was taken at 3, 12 and 24 hours. The
samples were serially diluted with 9 mL of pH 7.4 PBS. The cell
counts before and after gall treatment were estimated and also
shown in Table 4. It shows that these lactic acid bacteria remain
active in the environment mimicking the GI tract.
4 TABLE 4 Cell counts (Log CFU/mL) Before After treated with After
treated with Strain treatment HCl for 3 hours gall for 4 hours L.
plantarum 9.003 8.114 7.097 L. acidophilus 9.114 8.097 8.176 L.
casei 8.889 8.653 5.658 GM-080 9.029 7.699 6.602 L. bulgaricus
9.230 9.076 7.447
EXAMPLE 8
Animal Model
[0118] Animals. Female BALB/c mice were obtained from the National
Laboratory Animal Center in Taiwan and raised for 2 weeks in a room
where light and temperature were both controlled.
[0119] Allergen purification: The dust mite allergen, Der p 5, was
expressed in Escherichia coli comprising PGEX-2T expression vector
as a recombinant Der p 5-Glutathione S-transferase fusion protein
that can be purified with a glutathione-agarose binding
chromatography. The specific E. coli strain which is able to
express the desired allergen was cultured and induced. The bacteria
were collected and washed with TBS (pH 7.5) and added with 0.1 M
phenylmethylsulfony fluride. The cells were broken by adding DNase
I, Tween 20 and lysozyme, and by freeze-thaw method. The mixed
solution was added with EDTA and the residues were removed by
centrifugation to obtain the supernatant containing recombinant Der
p 5-Glutathione S-transferase fusion protein. The supernatant was
subjected to a glutathione-agarose affinity column for absorbing
the fusion protein. The column was then washed with TBS buffer at
4.degree. C. and then with reduced glutathion in Tris base (pH 8.0)
for separating the protein from column. The molecular weight of the
protein was estimated by SDS-PAGE and the concentration was also
assayed.
[0120] Sensitization: Mice were actively sensitized by
intraperitoneal injection of 10 .mu.g of Der p 5 with 4 mg of
aluminium hydroxide. 14 and 21 days after the initial
sensitization, the mice were exposed to an aerosol of 0.1% of the
purified Der p 5 for 30 min to perform inhalation challenge.
[0121] Treatment: The sensitized mice were divided into three
groups for the experiments. The mice of Group A were fed ten times
in two weeks with MRS broth as a control group. The mice of Group B
were administered with Lactobacillus casei ten times in two weeks
and 10.sup.9 CFU of bacteria were administered every time. Group B
was taken as a positive control, because L. casei had been
evidenced to be effective on inhibiting IgE secretion. The mice of
Group C were administered with GM-080 ten times in two weeks and
10.sup.9 CFU of bacteria were administered every time.
EXAMPLE 9
IgG and IgE Secretion
[0122] Determination of Der p 5-specific IgG and IgE: Eighteen
hours after last inhalation challenge, 500 .mu.L of blood sample
was taken from the tail. The blood samples were kept at room
temperature for 1 hour and then subjected to centrifugation. The
sera were stored at -80.degree. C. The amounts of Der p 5-specific
IgG2a, and IgE were determined by ELISA. Protein high-binding
plates with 96 wells were coated with 200 .mu.L of purified Der p 5
diluted in coating buffer (0.1 M NaHCO.sub.3, pH 9.6) at a
concentration of 10 .mu.g/mL. After overnight incubation at
4.degree. C., the plates were washed with PBS-Tween 20 and then
added with 300 .mu.L blocking buffer (3% BSA). After shaking for 2
hours at room temperature, the plates were washed again with
PBS-Tween 20. Sera were used at 1:10 dilution for IgG measurement
and 1:4 dilution for IgE measurement. The samples were shaken at
room temperature for 2 hours. After overnight incubation at
4.degree. C., the plates were washed with PBS-Tween 20, and added
with 200 .mu.L biotinylated rat anti-mouse IgE monoclonal antibody,
or rat anti-mouse IgG mAb. The sample was shaken at room
temperature for 2 hours and then washed with PBS-Tween 20. 200
.mu.L of Streptavidin-alkaline phosphatase (1:1000) was then added
and shaking the sample at room temperature for 1 h. After 6 washes,
color reaction was imitiated with the addition of 200 .mu.L
phosphatase substrate p-nitrophenyl phosphate, di-sodium salt
(pNPP) (Sigma.RTM. N-2770, USA). Plates were read in a microplate
autoreader (Metertech.RTM., Taiwan) at 405 nm.
[0123] Statistical analysis: To assay the changes of IgE and IgG
levels, repeated measures for analysis of One-way ANOVA were
performed to compare the differences between the groups. After
analysis of variance, Duncan multiple range tests were used to
differentiate differences between experimental and control groups.
A value of p<0.05 was used to indicate a statistically
significant difference.
[0124] Result. The result was shown in FIG. 7. It evidenced that
IgE secretion in sera of the animal treated with GM-080 was
enormously lowered and only 25% of that of without treatment. On
the other hand, IgG secretion in sera of the animal treated with
GM-080 was raised to two fold. Because IgG secretion represents Th1
T cell reaction, GM-080 is directed to eliminate IgE secretion that
correlated to allergy related disease.
EXAMPLE 10
Bronchoalveolar Lavage Fluid Cell Count
[0125] Samples preparation: Eighteens hours after sensitization,
the mice were lavaged with 5.times.0.5-ml aliquots of 0.9% sterile
saline through a polyethylene tube introduced through a
tracheostomy. Lavage fluid was centrifuged (500 g for 10 min at
4.degree. C.), and the cell pellet was resuspended in 1 ml of PBS
solution. Differentiated cell counts were made from cytospin
preparations stained by Leu's stain.
[0126] Statistical analysis: To assay the changes of cell counts,
repeated measures for analysis of One-way ANOVA were performed for
comparing the differences between the groups. After analysis of
variance, Duncan multiple range tests were used to differentiate
differences between experimental and control groups. A value of
p<0.05 was used to indicate a statistically significant
difference.
[0127] Result: The result was shown in FIG. 8. The blood cell type
contribution in the BALF represents the degrees of
inflammation.
[0128] Furthermore, the main symptoms of allergenic asthma are
chronic 5 inflammation in airway and eosinophils infiltration. It
evidenced that the eosinophils in the BALF of the animal treated
with GM-080 was enormously lowered from 5% to 1%. On the other
hand, the macrophages and lymphocytes in the BALF of the animal
treated with GM-080 were significantly raised.
EXAMPLE 11
IFN-.gamma. Secretion in The Bronchoalveolar Lavage Fluid
[0129] Samples preparation: After 24 hours of sensitization, the
mice were lavaged with 5.times.0.5-ml aliquots of 0.9% sterile
saline through a polyethylene tube introduced through a
tracheostomy. Lavage fluid was centrifuged (500 g for 10 min at
4.degree. C.), and the supernatant was subjected to IFN-.gamma.
quantitative analysis as described in Example 1.
[0130] Result: The result was shown in FIG. 9. It showed that the
animals fed with GM-080 produced about 100 pg/mL of IFN-.gamma. in
the BALF. On the other hand, the control group produced only 20 to
40 pg/mL of IFN-.gamma. in the BALF. GM-080 was effective on
inhibiting allergenic inflammation.
EXAMPLE 12
Inactive GM-080 for Treating Allergy
[0131] Inactive GM-080 preparation: Lyophilized GM-080 powder was
suspended in distilled water and autoclaved (121.degree. C., 15
min) before feeding mice.
[0132] Mice and sensitization: Female BALB/c mice (6-8 week-old)
were 25 purchased from National Laboratory Animal Breeding and
Research Center (Taipei, Taiwan). All animals were maintained
individually in cages with controlled temperature (24.+-.2.degree.
C.) and humidity (60.+-.5%) and maintained on a 12-h light-dark
cycle under specific-pathogen-free conditions. BALB/c mice were
i.p. with 10 g recombinant Dermatophagoides pteronyssinus allergen
Der p5-6.times. His fusion protein adsorbed to 4 mg of alumium
hydroxide. The mice were fed with 10.sup.7, 10.sup.9 and 10.sup.11
CFU GM-080 per mouse per day for three weeks. The mice were boosted
with the same dosage of allergen as sensitization at 14th day and
were challenged with 0.1% of Der p5-6.times. His diluted in PBS 21
days after sensitization. The inhalation challenge was performed in
1-L chamber connected to a DeVilbiss.TM. pulmosonic nebulizer
(Model 2512; DeVilbiss.RTM. Corp., Somerset, Pa.), which generated
an aerosol mist. After 18 hours, serum was collected by tail vein
bleeding and IgE was determined by ELISA as described in Example
9.
[0133] Result: The result was shown in FIG. 10. It showed that
BALB/c mice challenged with dust allergy Der p-5 had significantly
elevated serum IgE levels compared to naive group (p<0.05). It
suggested that allergic sensitized mice model could be successfully
set up. After feeding of different dosage of GM-080 per day for 21
days, the serum IgE in GM-080 group had significantly decreased
(p<0.05) compared with control group. The results showed that
inactive GM-080 could decrease the allergic responses by reducing
the allergen-specific IgE.
[0134] While embodiments of the present invention have been
illustrated and described, various modifications and improvements
can be made by persons skilled in the art. It is intended that the
present invention is not limited to the particular forms as
illustrated, and that all the modifications not departing from the
spirit and scope of the present invention are within the scope as
defined in the appended claims.
Sequence CWU 1
1
2 1 18 DNA Artificial L. paracasei specific 16S rRNA 1 caccgagatt
caacatgg 18 2 24 DNA Artificial conserved 16S rRNA 2 cccactgctg
cctcccgtag gagt 24
* * * * *
References